The programmed elimination of nuclei and organelles from the cytoplasm of differentiating lens fiber cells is one of the most characteristic features of lens cell differentiation. However, the molecular mechanisms underlying organelle degradation remain obscure. A better understanding of this process is necessary, because the abnormal retention of organelles is a feature of cataracts in both humans and animals. Organelle degradation involves the rapid removal of organelles and their proteinaceous contents. Consequently, there is considerable interest in identifying the proteolytic pathways that are activated during organelle degradation. In the first specific aim, we will test the hypothesis that the ubiquitin proteasome pathway (UPP) plays a critical role in the removal of organelles. These studies will utilize a novel in vivo chicken model in which the effects of selected proteasome inhibitors on organelle breakdown can be assessed directly. Recent studies have shown that degradation of fiber cell chromatin depends on the activity of DLAD (DNAse ll-like acid DNAase). We have obtained animals in which the gene for this nuclease has been knocked out. We will utilize these mice, in conjunction with transgenic animals expressing GFP- or His-tagged forms of DLAD, to understand how the DLAD nuclease gains access to the fiber cell chromatin and its mechanism of activation. Finally, mice in which the gene for Heat Shock Factor 4 (HSF4) has been deleted do not show the usual pattern of organelle loss. In these animals, intact organelles persist in the central lens fiber cells. We hypothesize that HSF4 regulates the expression of genes required for the initiation of organelle degradation. We will use a microarray-based strategy in wildtype and HSF4 null animals to identify such genes. Together these experiments will provide important new information on the mechanism(s) of organelle degradation in lens fiber cells and additional insight into the etiology of certain types of cataract.